1. Field of the Invention
This invention relates to production of materials for study as potential anthelmintic agents and as protective antigens.
2. Prior Art
Nematodes are parasites which are found in farm animals and in man and are the cause of disease therein.
Haemonchosis is a disease of ruminants due to the presence, in the gastro-intestinal tract, of the blood-feeding nematode Haemonchus. World-wide it is of very considerable economic importance having effects which range from reduction in weight gain, loss of production and agalactia through to death of domesticated animals. Ostertagiasis is caused by a related nematode Ostertagia and has similar effects. Both diseases are characterised by wasting due in part to anorexia associated with severe infections. In sheep and cattle, Ostertagia is the most important in winter rainfall areas, while Haemonchus is more important in summer rainfall zones. In Australia, for example, it is estimated that approximately one third of the 300 million sheep in the country are likely to be infected with Haemonchus.
The adult female of the nematode Haemonchus is up to 3 cm long and about 1 mm wide and has a diagonal red and white striped appearance. For this reason it is sometimes known as the barber's pole worm. The red colour is due to the presence of the host's blood in the intestine of the parasite. The parasitic stage of the worm lives attached to the wall of the stomach of the host animal, e.g. sheep, goat or cow, which it damages causing loss of blood in addition to that which it ingests. The animal becomes infected by ingesting infective larvae as it grazes on infested pastures. In the rumen the larva moults and passes into the abomasum where it develops, attaches itself to the stomach wall and commences ingesting blood. Over the following 7 days or so it continues to grow and then moults to give the young adult stage. About 18 to 21 days after infection it has become sexually mature and mates. The eggs pass out with the host faeces and hatch in due course into free-living larvae which develop into infective larvae.
Ostertagia differs from Haemonchus in that it feeds on the host animal's plasma, not on its blood. It has a smaller intestine than Haemonchus.
Although control of nematodes, such as Haemonchus and Ostertagia, can be achieved in sheep and cattle by administration of anthelmintic drugs, this approach is far from satisfactory as repeated dosing is necessary in order to achieve any degree of control over an infestation in a flock of sheep or herd of cattle.
Numerous attempts have been made to develop vaccines to Haemonchus and to Ostertagia including use of attenuated live worms and extracts of the worms. However, so far a commercially successful vaccine has not yet been produced. Although injection of extracts of various kinds produced from Haemonchus or Ostertagia has in some cases led to generation of antibodies directed against the parasite, such antibodies appear to be similar to those formed by natural infection and do not seem to enhance natural resistance to the parasite.
I have previously reported in "Tissue & Cell", 1977, 9 (1) at pages 23 to 34, the existence of a helical, polymeric extracellular protein which is associated with the luminal surface of Haemonchus contortus intestinal cells. In the fourth stage larvae and adult nematodes (the parasitic stages) the microvilli on the intestinal cells of Haemonchus contortus have associated therewith filaments in the form of helices about 400.ANG. in diameter and of variable pitch. These filaments proved to be primarily protein which I designated as contortin. On page 24 of the paper cited above, "Tissue & Cell" 1977 9(1), pages 23-34, a process for preparing contortin is described. In this process, approximately 2 g net weight adult Haemonchus contortus were homogenized in 60 ml 10 nM phosphate buffered saline (pH 7.3). The homogenate was centrifuged for 5 minutes at 3000 rpm in a 6450 rotor of an MSE Minor centrifuge at 4.degree. C. The supernatant was decanted and the pellets homogenized again and the homogenate re-centrifuged. The pooled supernatants were centrifuged at 10000 g for 10 minutes in a SS-34 rotor of a Sorvall RC-2 refrigerated centrifuge. The pellets were washed once on the centrifuge and the pooled supernatants centrifuged for 90 minutes at 50000 rpm in a Type 50 rotor of a Bechman Spinco Model L centrifuge. The sedimented material contained the bulk of the contortin in its extracellular form as judged by electron microscopy of negatively stained samples.
Further investigation showed that the helical filament of contortin appears to be formed by lateral polymerization of a monomeric form which has a Y-shaped structure with arms about 45 .ANG. long and 25 .ANG. wide seen in negatively stained preparations. A further description of contortin appeared in "Proceedings of the Sixth European Congress on Electron Microscopy", Vol II, pages 515 and 516. Ostertagia circumcincta also has contortin-like material associated with the microvilli in its intestine.
A brief report that antibodies against contortin have been raised in rabbits with a view to testing whether contortin is antigenic and would act as a vaccine in sheep appeared in the "Report for 1974-75" of the Agricultural Research Council Institute of Animal Physiology, Babraham, in June 1976. In the corresponding "Report for 1976-77" published in 1978, there were summarised on page 94 the results of furthest work by me in which I showed that intramuscular injection of contortin-enriched fractions into young lambs gave a substantial measure of protection against haemonchosis when they were subsequently challenged with larvae of Haemonchus contortus. This work on the use of a contortin-enriched preparation for protection against haemonchosis has been published in "Parasitology", 94, at pages 385 to 397.
Although a contortin-enriched fraction can be used to protect lambs against infection by Haemonchus contortus, such a fraction is not commercially useful as it has generally required injection of a large quantity of such a fraction in order to produce in the animal a suitable immune response. Moreover it is difficult to obtain either the polymeric or monomeric form of contortin in purer form and in sufficient quantity to facilitate further study of their antigenic characteristics.
There is also present in the supernatant liquor from which contortin is obtained by the procedure described in the afore-mentioned article in "Tissue & Cell" and "Parasitology", a further material which appears to be primarily protein, which has a molecular weight of approximately 61,000, and which reacts with antibodies to contortin. This protein I have named pre-contortin.